Protocol S1.
Inhibition of mutation and combating the evolution of antibiotic resistance
Ryan T. Cirz, Jodie K. Chin, David R. Andes, Valérie de Crécy-Lagard,
William A. Craig and Floyd E. Romesberg
Strain Construction. Strains were constructed by first generating a disruption cassette
using 3-way PCR as described by Murphy [1].
Deletion cassettes consisted of
approximately 500 bp regions upstream and downstream of the gene to be deleted,
including the first and last 2-10 codons of the gene, flanking an antibiotic resistance
cassette in reverse orientation. E. coli specific sequences were amplified from MG1655
genomic DNA and purified with the DNeasy Tissue kit (Qiagen). The KmR cassette was
amplified from pUC4K [2], the SpecR cassette from pOmega [3], and the CmR cassette
from pSU18 [4]. Oligonucleotide primers used in the construction of the disruption
cassettes are listed in Table S1.
The lexA(S119A) mutation cassette was constructed by a similar method.
Approximately 500 bp of sequence upstream of the lexA coding sequence and the
complete coding region of lexA was PCR-amplified from genomic MG1655 DNA using
the primers lexA_NF-SphI and lexA_OrfR-NdeI, digested with SphI/NdeI and ligated
into SphI/NdeI digested pUC18 vector [5].
The S119A mutation (TCG–>GCG) was
introduced in the resulting plasmid using the Quikchange Site-directed Mutagenesis kit
Protocol S1, Page 1
(Stratagene) with the primers LexA_S119A_QCF and LexA_S119A_QCR. The resulting
allele was confirmed by sequencing. A 500 bp fragment corresponding to the genomic
sequence downstream of lexA was amplified with the primers lexA_CF and lexA_CR.
The KmR cassette was amplified from pUC4K using the primers KanF and KanR. The
500 bp downstream fragment was PCR assembled with the Kan cassette using the 20 bp
homology intrinsic in primer lexA_CF (See Table S1). The pUC18 vector containing 500
bp of upstream DNA and the lexA(S119A) coding sequence was digested with SphI/NdeI
and the Kan cassette/downstream fragment assembly product was digested with NdeI.
The two products were ligated together and PCR amplified using the primers lexA_NFSphI and lexA_CR. The final lexA cassette consisted of 500 bp of upstream DNA, the
mutated lexA ORF, an NdeI site attaching a KmR marker in reverse orientation, and
approximately 500 bp of downstream DNA (see below).
STOP+10 base pair
Upstream/mutated lexA ORF
KmR (reverse)
Downstream of lexA STOP
NdeI site
lexA(S119A):KmR cassette
Figure S1. lexA(S119A):KmR cassette
The disruption cassettes were transformed by electroporation into strain PS6275,
plated on LB supplemented with the appropriate antibiotic, and grown at 30 C. After
confirmation of correct chromosomal insertion by PCR, cassettes were transferred into
MG1655 by P1 transduction [6] and confirmed by plating on minimal media lacking
biotin and containing the antibiotic used for cassette selection. Correct disruption was
Protocol S1, Page 2
confirmed by PCR; the lexA(S119A) strain was confirmed by PCR followed by
sequencing.
To construct the ATCC 25922 derivatives of the ∆lacZ and lexA(S119A) mutants,
the respective cassettes were transferred by P1 transduction from strains RTC0001 and
RTC0011 into ATCC 25922; colonies were selected on LB supplemented with 50 g/ml
kanamycin.
Transfer of the mutant alleles was confirmed by PCR followed by
sequencing for the lexA(S119A) mutant.
The gyrA mutant clones from different genetic backgrounds described in Table 2
and Table S2 were isolated during mutation assays. Because no gyrA mutant clones
could be isolated in the ∆recG, ∆ruvB, and ∆ruvC backgrounds, the three knockout
cassettes were each moved by P1 transduction into RTC0110 to create strains RTC0131,
RTC0132, and RTC0133, respectively (see Table 1 and Table 2).
Growth Rate Determination. For each strain, two independent cultures were grown for
25 h at 37 C in LB containing no antibiotic. These cultures were diluted 1:500 in LB and
grown at 37 °C with shaking. At each time point, the A600 was measured and cultures
were diluted and plated on LB/agar to determine the number of viable cells. Growth
from 90-300 was used to determine the doubling times for each strain (Table 2).
Minimum Inhibitory Concentration Determination. For each strain, two independent
cultures were grown for 25 h at 37 C in LB containing no antibiotic. From each culture,
~104–105 colony forming units (cfu) were used to inoculate, in duplicate (total of 4 data
points per strain), LB containing 12 different concentrations (0, 1.0, 3.0, 5.0, 7.5, 10, 15, 20,
25, 30, 35, 40, 50 ng/ml) of ciprofloxacin using 96-well flat bottom plates. After 18 h of
incubation at 37 C, growth was measured by reading A650 in a Vmax Kinetic Microplate
Protocol S1, Page 3
Reader (Molecular Devices, CA). The MIC was defined as the lowest concentration of
ciprofloxacin that prevented any detectable growth.
MICs for all strains were
determined in at least three independent experiments (total of 12 data points per strain
per ciprofloxacin concentration). MICs for ciprofloxacin-resistant clones isolated during
the mutation assays were measured with the same conditions used for the non-resistant
strains described above, except wells contained LB plus ciprofloxacin at 0, 50, 100, 150,
200, 250, 300, 350, 400, 500, 600, 800, 1000 ng/ml.
Sequencing.
Colonies from the reconstruction assays were streaked on LB/agar
containing 40 ng/ml ciprofloxacin. A single colony from each plate was used as a
colony PCR template for gyrA gene fragment amplification with the primers gyrA_OrfF
and gyrA_1KbR (Table S1). This region of the gyrA gene (base pairs 1-1000) contains the
quinolone resistance determining region (QRDR) [7], the most consistent location of
ciprofloxacin resistance mutations in gram negative bacteria. The PCR products were
purified using the Qiaquick PCR Purification kit (Qiagen) and sequenced with the
primers gyrA_OrfF and gyrA_Seq (Table S1). The number of mutants sequenced per
strain is listed in Table 3. Some ciprofloxacin-resistant clones had no mutations in the
gyrA region sequenced. MICs of these WT gyrA clones were always significantly lower
than any clones containing gyrA mutations (generally < 80-150 ng/ml, Table S2). These
mutant clones most likely contain mutations in the gyrB, parC, parE, marR, or acrR gene
or MarR or AcrR operator sites which could permit growth in the presence of a WT gyrA
under the conditions used (ciprofloxacin at 40 ng/ml) [8].
‘Second-Step’ Mutation and Survival Assays.
A ∆lacZ, gyrA(S83L) mutant and a
lexA(S119A), gyrA(S83L) mutant were both isolated during mutation assays with 40
Protocol S1, Page 4
ng/ml ciprofloxacin. These mutants were isolated 24 h after plating onto LB/agar plus
ciprofloxacin, and thus most likely were resistant prior to plating and exposure to the
drug. As a result, these mutants are not likely to have additional second-site mutations.
We examined the two clones in mutation, survival, and reconstruction assays as
described in the Methods section, with the exception of using media containing 650
ng/ml ciprofloxacin instead of 40 ng/ml.
Survival Assay Validation. To validate the accuracy of the survival assay (see Methods),
an additional experiment was performed. Three independent cultures of MG1655 were
grown overnight at 37 °C. From these cultures, 4 different volumes were plated on
minimal media/agar lacking a carbon source (M9 + 1 mM MgSO4) and incubated for 1 h
at 37 C. After incubation, plates were homogenized in saline, and the number of cells
on each M9 plate was determined (‘Calculated’, Table S3) in a manner analogous to the
survival assay. The actual number of cells plated (‘Actual’, Table S3) was determined by
plating serial dilutions of the overnight cultures onto LB/agar.
In all cases, the
calculated number of viable cells on the M9 plates was within ±2-fold of the actual
number of cells plated (Table S3).
Additional Reconstruction Assay. Our reconstruction assays (see Methods section)
were performed in the absence of competing, ciprofloxacin-sensitive cells. Although our
mutation assay differs significantly from other systems, the presence of competing cells
has affected colony regrowth time in other mutation assays [9]. To address this issue
and more accurately re-create the original conditions in which resistant colonies arose in
the mutation assay, an additional reconstruction assay was performed for each knockout
background/gyrA mutant combination isolated during mutation assays. Approximately
Protocol S1, Page 5
100 cfu of the gyrA mutant strain were plated on 40 ng/ml ciprofloxacin in the presence
of ~108 cells with the same genetic background, but with a wild-type gyrA gene. In all
cases, colonies from the resistant population arose in the same amount of time as in the
reconstruction experiments which lacked competing, ciprofloxacin sensitive cells.
Growth of gyrA(S83L) Mutants in Different Genetic Backgrounds. For each genetic
background that could carry a gyrA(S83L) mutation, the ciprofloxacin MIC was
measured (Table 2). Strains that have similar MICs should grow similarly on solid
media containing ciprofloxacin. However, in order to directly demonstrate this, the
∆lacZ gyrA(S83L), lexA(S119A) gyrA(S83L) and ∆polB ∆dinB ∆umuDC gyrA(S83L)
mutants were each grown overnight in permissive liquid culture and for each strain
approximately 100 cfu were plated on LB/agar containing 40 ng/ml ciprofloxacin (the
same concentration used in the mutation assay). After incubation for 24 h at 37 °C, the
plates all contained substantial, similar sized colonies (Figure S4).
References
1. Murphy KC, Campellone KG, Poteete AR. (2000) PCR-mediated gene replacement in
Escherchia coli. Gene 246:321-330.
2. Muller W, Keppner W, Rasched I. (1986) Versatile kanamycin-resistance cartridges
for vector construction in Escherichia coli. Gene 46:131-133.
3. Prentki P, Krisch HM. (1984) In vitro insertional mutagenesis with a selectable DNA
fragment. Gene 29:303-313.
Protocol S1, Page 6
4. Bartolome B, Jubete Y, Martinez E, Cruz F-d-l. (1991) Construction and properties of
a family of pACYC184-derived cloning vectors compatible with pBR322 and its
derivatives. Gene 102:75-78.
5. Vieira J, Messing J. (1982) The pUC plasmids, an M13mp7-derived system for
insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259268.
6. Miller JH. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory
Press.
7. Yoshida H, Bogaki M, Nakamura S, Ubukata K, Konno M. (1990) Nucleotidesequence and characterization of the Staphylococcus aureus norA gene, which confers
resistance to quinolones. J. Bacteriol. 172:6942-6949.
8. Lindgren PK, Karlsson Å, Hughes D. (2003) Mutation rate and evolution of
fluoroquinolone resistance in Escherichia coli isolates from patients with urinary tract
infections. Antimicrob. Agents Chemother. 47:3222-3232.
9. McKenzie GJ, Lombardo M-J, Rosenberg SM. (1998) Recombination-dependent
mutation in Escherichia coli occurs in stationary phase. Genetics 149:1163-1165.
Protocol S1, Page 7